Structure-activity analysis suggests an olfactory function for the unique antennal delta glutathione transferase of Apis mellifera.

Glutathione transferases (GST) are detoxification enzymes that conjugate glutathione to a wide array of molecules. In the honey bee Apis mellifera, AmGSTD1 is the sole member of the delta class of GSTs, with expression in antennae. Here, we structurally and biochemically characterized AmGSTD1 to elucidate its function. We showed that AmGSTD1 can efficiently catalyse the glutathione conjugation of classical GST substrates. Additionally, AmGSTD1 exhibits binding properties with a range of odorant compounds. AmGSTD1 has a peculiar interface with a structural motif we propose to call 'sulfur sandwich'. This motif consists of a cysteine disulfide bridge sandwiched between the sulfur atoms of two methionine residues and is stabilized by CH…S hydrogen bonds and S…S sigma-hole interactions. Thermal stability studies confirmed that this motif is important for AmGSTD1 stability and, thus, could facilitate its functions in olfaction.

The impact of temperature on insecticide sensitivity depends on transgenerational effects.

The combined effects of insecticides and temperature are increasingly being studied because species are expected to change their responses to insecticides with climate warming. As recently highlighted, the impact of temperature on insecticide sensitivity might be influenced by the environment experienced by the previous generation. However, a pioneering study that showed this transgenerational effect in the mosquito Culex pipiens needs to be confirmed because two other studies did not show similar results. Here, we performed an experiment on the moth Spodoptera littoralis to test this hypothesis. We analysed reaction norms among experimental families to test transgenerational effects, i.e., the variation in the response of families to the combined effects of the insecticide chlorpyrifos and developmental temperature. Reaction norm analyses revealed that the responses of the families to chlorpyrifos and temperature differed for developmental time and larval survival, two key parameters in S. littoralis. Crucially, for larval survival, a family effect influenced the impact of temperature on chlorpyrifos sensitivity. This finding confirms the pioneering study on C. pipiens that showed transgenerational effects on the combined effects of insecticides and temperature. This result also highlights that transgenerational plasticity can be important to consider in ecotoxicology.

Combined influences of transgenerational effects, temperature and insecticide on the moth Spodoptera littoralis.

Climate warming is expected to impact the response of species to insecticides. Recent studies show that this interaction between insecticides and temperature can depend on other factors. Here, we tested for the influence of transgenerational effects on the Insecticide × Temperature interaction in the crop pest moth Spodoptera littoralis. Specifically, we analysed reaction norms among experimental clutches based on a split-plot design crossing the factors temperature, insecticide and clutch. The study was performed on 2280 larvae reared at four temperatures (23, 25, 27 and 29 °C), and their response to the insecticide deltamethrin (three concentrations and a control group) was tested. Temperature had a global influence with effects on larval survival, duration of development, pupal body mass, and significant reaction norms of the clutches for temperature variations of only 2 °C. In addition to the expected effect of deltamethrin on mortality, the insecticide slightly delayed the development of S. littoralis, and the effects on mortality and development differed among the clutches. Projection models integrating all the observed responses illustrated the additive effects of deltamethrin and temperature on the population multiplication rate. Variation in the response of the clutches showed that transgenerational effects influenced the impact of insecticide and temperature. Although no evidence indicated that the Insecticide × Temperature interaction depended on transgenerational effects, the studies on the dependence of the Insecticide × Temperature interaction on other factors continue to be crucial to confidently predict the combined effects of insecticides and climate warming.

Resistance in the Genus Spodoptera: Key Insect Detoxification Genes.

The genus Spodoptera (Lepidoptera: Noctuidae) includes species that are among the most important crop pests in the world. These polyphagous species are able to feed on many plants, including corn, rice and cotton. In addition to their ability to adapt to toxic compounds produced by plants, they have developed resistance to the chemical insecticides used for their control. One of the main mechanisms developed by insects to become resistant involves detoxification enzymes. In this review, we illustrate some examples of the role of major families of detoxification enzymes such as cytochromes P450, carboxyl/cholinesterases, glutathione S-transferases (GST) and transporters such as ATP-binding cassette (ABC) transporters in insecticide resistance. We compare available data for four species, Spodoptera exigua, S. frugiperda, S. littoralis and S. litura. Molecular mechanisms underlying the involvement of these genes in resistance will be described, including the duplication of the CYP9A cluster, over-expression of GST epsilon or point mutations in acetylcholinesterase and ABCC2. This review is not intended to be exhaustive but to highlight the key roles of certain genes.

Detoxification gene families in Phylloxera: Endogenous functions and roles in response to the environment.

Phylloxera, Daktulosphaira vitifoliae, is an agronomic pest that feeds monophagously on grapevine, Vitis spp. host plants. Phylloxera manipulates primary and secondary plant metabolism to establish either leaf or root galls. We manually annotated 198 detoxification genes potentially involved in plant host manipulation, including cytochrome P450 (66 CYPs), carboxylesterase (20 CCEs), glutathione-S-transferase (10 GSTs), uridine diphosphate-glycosyltransferase (35 UGTs) and ABC transporter (67 ABCs) families. Transcriptomic expression patterns of these detoxification genes were analyzed for root and leaf galls. In addition to these transcriptomic analyses, we reanalyzed recent data from L1 and L2-3 stages feeding on tolerant and resistant rootstock. Data from two agricultural pest aphids, the generalist Myzus persicae and the Fabaceae specialist Acyrthosiphon pisum, and from the true bug vector of Chagas disease, Rhodnius prolixus, were used to perform phylogenetic analyses for each detoxification gene family. We found expansions of several gene sub-families in the genome of D. vitifoliae. Phylogenetically close genes were found to be organized in clusters in the same genomic position and orientation suggesting recent successive duplications. These results highlight the roles of the phylloxera detoxification gene repertoire in insect physiology and in adaptation to plant secondary metabolites, and provide gene candidates for further functional analyses.

Sublethal Exposure Effects of the Neonicotinoid Clothianidin Strongly Modify the Brain Transcriptome and Proteome in the Male Moth Agrotis ipsilon.

Insect pest management relies mainly on neurotoxic insecticides, including neonicotinoids such as clothianidin. The residual accumulation of low concentrations of these insecticides can have positive effects on target pest insects by enhancing various life traits. Because pest insects often rely on sex pheromones for reproduction and olfactory synaptic transmission is cholinergic, neonicotinoid residues could indeed modify chemical communication. We recently showed that treatments with low doses of clothianidin could induce hormetic effects on behavioral and neuronal sex pheromone responses in the male moth, Agrotis ipsilon. In this study, we used high-throughput RNAseq and proteomic analyses from brains of A. ipsilon males that were intoxicated with a low dose of clothianidin to investigate the molecular mechanisms leading to the observed hormetic effect. Our results showed that clothianidin induced significant changes in transcript levels and protein quantity in the brain of treated moths: 1229 genes and 49 proteins were differentially expressed upon clothianidin exposure. In particular, our analyses highlighted a regulation in numerous enzymes as a possible detoxification response to the insecticide and also numerous changes in neuronal processes, which could act as a form of acclimatization to the insecticide-contaminated environment, both leading to enhanced neuronal and behavioral responses to sex pheromone.

Chromosomal scale assembly of parasitic wasp genome reveals symbiotic virus colonization.

Endogenous viruses form an important proportion of eukaryote genomes and a source of novel functions. How large DNA viruses integrated into a genome evolve when they confer a benefit to their host, however, remains unknown. Bracoviruses are essential for the parasitism success of parasitoid wasps, into whose genomes they integrated ~103 million years ago. Here we show, from the assembly of a parasitoid wasp genome at a chromosomal scale, that bracovirus genes colonized all ten chromosomes of Cotesia congregata. Most form clusters of genes involved in particle production or parasitism success. Genomic comparison with another wasp, Microplitis demolitor, revealed that these clusters were already established ~53 mya and thus belong to remarkably stable genomic structures, the architectures of which are evolutionary constrained. Transcriptomic analyses highlight temporal synchronization of viral gene expression without resulting in immune gene induction, suggesting that no conflicts remain between ancient symbiotic partners when benefits to them converge.

Correction to: The genome sequence of the grape phylloxera provides insights into the evolution, adaptation, and invasion routes of an iconic pest.

An amendment to this paper has been published and can be accessed via the original article.

A maternal effect influences sensitivity to chlorpyrifos pesticide in the pest moth Spodoptera littoralis.

Transgenerational effects on sensitivity to pesticides are poorly studied. This study investigated the transgenerational influences of maternal body mass in the major pest moth Spodoptera littoralis, with a focus on sensitivity to chlorpyrifos pesticide. In 147 clutches of a laboratory strain of S. littoralis, we compared larval mortality between control larvae and larvae treated with chlorpyrifos. Because of the classic positive relationships between offspring size and maternal size and between offspring size and offspring quality, sensitivity to chlorpyrifos was predicted to be lower in larvae of larger mothers. Surprisingly, we found the opposite result, with higher pesticide toxicity in larvae of larger mothers. This result is partly explained by the lack of a relationship between larval mass and larval sensitivity to chlorpyrifos. This means that another offspring characteristic linked to maternal size should have affected larval sensitivity to chlorpyrifos. More generally, knowledge of the effects of the traits and ecological environments of mothers on offspring sensitivity to pesticides remains limited. Ecotoxicologists should pay more attention to such maternal effects on sensitivity to pesticides, both in pests and non-target species.

Effects of low concentrations of deltamethrin are dependent on developmental stages and sexes in the pest moth Spodoptera littoralis.

Effects of low concentrations of pesticides, with no or moderate mortality of targeted species, are poorly studied even though these low concentrations are common under natural conditions. Studying their effects is critical because they can induce positive hormetic responses, possibly leading to greater pest multiplication and promoting the evolution of pest resistance. Here, we investigated the responses of the pest moth Spodoptera littoralis to low concentrations of deltamethrin, and tested for variation in effects of the pesticide between developmental stages and sexes. Indeed, we show that a given concentration of deltamethrin has different effects between stages, and even between sexes. Two experimental concentrations led to very high mortality early in S. littoralis development (4th larval instar), but only to low mortality rates in adults. Moreover, our highest experimental concentration had only detrimental effects in adult females, but improved the reproductive success of adult males. Model projections showed that the lethality from treatments at the 4th larval instar was the predominant effect. Because of the high multiplication rate of S. littoralis, it was also found that treatments with very similar effects on larval mortality can lead to either population extinction or rapid pest resurgence.

The genome sequence of the grape phylloxera provides insights into the evolution, adaptation, and invasion routes of an iconic pest.

BACKGROUND: Although native to North America, the invasion of the aphid-like grape phylloxera Daktulosphaira vitifoliae across the globe altered the course of grape cultivation. For the past 150 years, viticulture relied on grafting-resistant North American Vitis species as rootstocks, thereby limiting genetic stocks tolerant to other stressors such as pathogens and climate change. Limited understanding of the insect genetics resulted in successive outbreaks across the globe when rootstocks failed. Here we report the 294-Mb genome of D. vitifoliae as a basic tool to understand host plant manipulation, nutritional endosymbiosis, and enhance global viticulture. RESULTS: Using a combination of genome, RNA, and population resequencing, we found grape phylloxera showed high duplication rates since its common ancestor with aphids, but similarity in most metabolic genes, despite lacking obligate nutritional symbioses and feeding from parenchyma. Similarly, no enrichment occurred in development genes in relation to viviparity. However, phylloxera evolved > 2700 unique genes that resemble putative effectors and are active during feeding. Population sequencing revealed the global invasion began from the upper Mississippi River in North America, spread to Europe and from there to the rest of the world. CONCLUSIONS: The grape phylloxera genome reveals genetic architecture relative to the evolution of nutritional endosymbiosis, viviparity, and herbivory. The extraordinary expansion in effector genes also suggests novel adaptations to plant feeding and how insects induce complex plant phenotypes, for instance galls. Finally, our understanding of the origin of this invasive species and its genome provide genetics resources to alleviate rootstock bottlenecks restricting the advancement of viticulture.

Effects of DEHP on the ecdysteroid pathway, sexual behavior and offspring of the moth Spodoptera littoralis.

Bis(2-ethylhexyl) phthalate (DEHP) is a widely produced plasticizer that is considered to act as an endocrine-disrupting chemical in vertebrates and invertebrates. Indeed, many studies have shown that DEHP alters hormonal levels, reproduction and behavior in vertebrates. Few studies have focused on the effects of DEHP on insects, although DEHP is found almost everywhere in their natural habitats, particularly in soils and plants. Here, we investigated the effects of DEHP on the sexual behavior and physiology of a pest insect, the noctuid moth Spodoptera littoralis. In this nocturnal species, olfaction is crucial for sexual behavior, and ecdysteroids at the antennal level have been shown to modulate sex pheromone detection by males. In the present study, larvae were fed food containing different DEHP concentrations, and DEHP concentrations were then measured in the adults (males and females). Hemolymphatic ecdysteroid concentrations, the antennal expression of genes involved in the ecdysteroid pathway (nuclear receptors EcR, USP, E75, and E78 and calmodulin) and sexual behavior were then investigated in adult males. The success and latency of mating as well as the hatching success were also studied in pairs consisting of one DEHP male and one uncontaminated female or one DEHP female and one uncontaminated male. We also studied the offspring produced from pairs involving contaminated females to test the transgenerational effect of DEHP. Our results showed the general downregulation of nuclear receptors and calmodulin gene expression associated with the higher concentrations of DEHP, suggesting peripheral olfactory disruption. We found some effects on male behavior but without an alteration of the mating rate. Effects on offspring mortality and developmental rates in the N + 1 generation were also found at the higher doses of DEHP. Taken together, the results of the study show for the first time that larval exposure to DEHP can induce delayed endocrine-disruptive effects in the adults of a terrestrial insect as well as effects on the next generation. To date, our study is also the first description of an impact of endocrine disrupter on olfaction in insects.

The Drosophila odorant-binding protein 28a is involved in the detection of the floral odour ß-ionone.

Odorant-binding proteins (OBPs) are small soluble proteins that are thought to transport hydrophobic odorants across the aqueous sensillar lymph to olfactory receptors. A recent study revealed that OBP28a, one of the most abundant Drosophila OBPs, is not required for odorant transport, but acts in buffering rapid odour variation in the odorant environment. To further unravel and decipher its functional role, we expressed recombinant OBP28a and characterized its binding specificity. Using a fluorescent binding assay, we found that OBP28a binds a restricted number of floral-like chemicals, including ß-ionone, with an affinity in the micromolar range. We solved the X-ray crystal structure of OBP28a, which showed extensive conformation changes upon ligand binding. Mutant flies genetically deleted for the OBP28a gene showed altered responses to ß-ionone at a given concentration range, supporting its essential role in the detection of specific compounds present in the natural environment of the fly.

Effects of bisphenol A on post-embryonic development of the cotton pest Spodoptera littoralis.

Endocrine-disrupting chemicals encompass a variety of chemicals that may interfere with the endocrine system and produce negative effects on organisms. Among them, bisphenol A is considered a major pollutant in numerous countries. The harmful effects of BPA on environmental and human health are intensely studied. However, the effects of BPA on terrestrial insects are still poorly investigated, despite that several plants can accumulate BPA in their tissues, leading to potential contamination of herbivorous insects. Here, we used the leafworm Spodoptera littoralis, a polyphagous species, to study BPA effects on post-embryonic development. We studied the effects of BPA ingestion at environmental doses (e.g., 0.01, 0.1, and 1 µg/g of BPA) and high doses (e.g., 25 µg/g) on larval weight and stage duration, pupal length and sex ratio. BPA effects were investigated in more detail during the last larval instar, a crucial period for preparing pupation and metamorphosis, which are under endocrine control. We monitored the haemolymph concentration of ecdysteroids, hormones controlling moult and metamorphosis, as well as the expression levels of several nuclear receptors involved in the ecdysteroid signalling pathway. Our integrative study showed that, upon exposure doses, BPA can induce various effects on the viability, developmental time, growth and sex ratio. These effects were correlated with a delay of the ecdysteroid peak during the last larval instar and a modification of expression of EcR, USP, E75AB, E75D and Br-c. We provide new evidence about the events that occur after BPA exposure in insect contaminated by food ingestion.

Effects of DEHP on post-embryonic development, nuclear receptor expression, metabolite and ecdysteroid concentrations of the moth Spodoptera littoralis.

Di (2-ethylhexyl) phthalate (DEHP) is recognized in vertebrates as an Endocrine Disrupting Chemical (EDC). DEHP can alter steroid hormones production, development, reproduction and behavior in vertebrates. Only few studies investigated DEHP effects on insects. However, some recent studies on aquatic insects showed that DEHP could also act as an EDC by interfering with the signaling pathways of ecdysteroids, the main hormones involved in the control of insect post-embryonic development and physiology. The aim of the study was to investigate (1) the fate of DEHP within a terrestrial insect species by exposing larvae to food containing a wide range of DEHP concentrations and (2) the effects of this chemical on their post-embryonic development and metamorphosis, by using a multi-level approach. DEHP was shown to be present both in larvae and resulting stages, with higher concentrations in chrysalises and adults than in larvae. DEHP concentrations also decreased at the end of the last larval instar, suggesting the metabolic transformation or excretion of this chemical during this time. Only the two highest DEHP doses induced higher insect mortality, whereas low and intermediate concentrations increased larval food consumption without affecting body weight. Metabolic profiles showed that in control insects, the last three days before metamorphosis correspond to a metabolic transition, but with time-dependent changes in treated insects. Interestingly, DEHP treatments also alter both hemolymphatic ecdysteroid titers and expression levels of ecdysteroid response genes. These results confirm that DEHP can alter insect post-embryonic development and metamorphosis, by interfering with ecdysteroid pathways.

Glutathione-S-Transferases in the Olfactory Organ of the Noctuid Moth Spodoptera littoralis, Diversity and Conservation of Chemosensory Clades.

Glutathione-S-transferases (GSTs) are conjugating enzymes involved in the detoxification of a wide range of xenobiotic compounds. The expression of GSTs as well as their activities have been also highlighted in the olfactory organs of several species, including insects, where they could play a role in the signal termination and in odorant clearance. Using a transcriptomic approach, we identified 33 putative GSTs expressed in the antennae of the cotton leafworm Spodoptera littoralis. We established their expression patterns and revealed four olfactory-enriched genes in adults. In order to investigate the evolution of antennal GST repertoires in moths, we re-annotated antennal transcripts corresponding to GSTs in two moth and one coleopteran species. We performed a large phylogenetic analysis that revealed an unsuspected structural-and potentially functional-diversity of GSTs within the olfactory organ of insects. This led us to identify a conserved clade containing most of the already identified antennal-specific and antennal-enriched GSTs from moths. In addition, for all the sequences from this clade, we were able to identify a signal peptide, which is an unusual structural feature for GSTs. Taken together, these data highlight the diversity and evolution of GSTs in the olfactory organ of a pest species and more generally in the olfactory system of moths, and also the conservation of putative extracellular members across multiple insect orders.

Neofunctionalization of "Juvenile Hormone Esterase Duplication" in Drosophila as an odorant-degrading enzyme towards food odorants.

Odorant degrading enzymes (ODEs) are thought to be responsible, at least in part, for olfactory signal termination in the chemosensory system by rapid degradation of odorants in the vicinity of the receptors. A carboxylesterase, specifically expressed in Drosophila antennae, called "juvenile hormone esterase duplication (JHEdup)" has been previously reported to hydrolyse different fruit esters in vitro. Here we functionally characterize JHEdup in vivo. We show that the jhedup gene is highly expressed in large basiconic sensilla that have been reported to detect several food esters. An electrophysiological analysis demonstrates that ab1A olfactory neurons of jhedup mutant flies exhibit an increased response to certain food acetates. Furthermore, mutant flies show a higher sensitivity towards the same odorants in behavioural assays. A phylogenetic analysis reveals that jhedup arose as a duplication of the juvenile hormone esterase gene during the evolution of Diptera, most likely in the ancestor of Schizophora, and has been conserved in all the 12 sequenced Drosophila species. Jhedup exhibits also an olfactory-predominant expression pattern in other Drosophila species. Our results support the implication of JHEdup in the degradation of food odorants in D. melanogaster and propose a neofunctionalization of this enzyme as a bona fide ODE in Drosophilids.

Molecular basis for the behavioral effects of the odorant degrading enzyme Esterase 6 in Drosophila.

Previous electrophysiological and behavioural studies implicate esterase 6 in the processing of the pheromone cis-vaccenyl acetate and various food odorants that affect aggregation and reproductive behaviours. Here we show esterase 6 has relatively high activity against many of the short-mid chain food esters, but negligible activity against cis-vaccenyl acetate. The crystal structure of esterase 6 confirms its substrate-binding site can accommodate many short-mid chain food esters but not cis-vaccenyl acetate. Immunohistochemical assays show esterase 6 is expressed in non-neuronal cells in the third antennal segment that could be accessory or epidermal cells surrounding numerous olfactory sensilla, including basiconics involved in food odorant detection. Esterase 6 is also produced in trichoid sensilla, but not in the same cell types as the cis-vaccenyl acetate binding protein LUSH. Our data support a model in which esterase 6 acts as a direct odorant degrading enzyme for many bioactive food esters, but not cis-vaccenyl acetate.